Semen is a complex organic fluid that contains sperm and seminal fluid proteins (SFPs), the latter of which are produced by different secretory tissues of the male reproductive tract. In insects, hundreds of SFPs have been identified over the last few years. While little is known about their pre-mating functions in males, in females some SFPs have been shown to induce physiological and behavioural changes after mating, including changes in the willingness to re-mate, egg production rates, sperm storage, innate immunity, as well as flight and feeding behaviour. A recent study by a US team of researchers led by Reddy Palli, from the University of Kentucky, focused on identifying SFPs that are transferred from the male to the female in the red flour beetle, Tribolium castaneum, in the hope of identifying their molecular role in female reproduction.
To analyse SFP function, the team chose T. castaneum as a model, because of its fully sequenced genome and its amenability to systemic RNA interference (RNAi), a method that allows quick and efficient knock-down of gene expression. In a previous microarray study, Palli's team had identified 112 SFP genes from T. castaneum. However, it was unclear which SFPs are actually transferred to the females' sperm storage organ, the spermatheca. To identify SFPs that are transferred during mating, the scientists analysed protein extracts from seminal vesicles and spermathecae by mass spectrometry, as well as comparing the relative gene transcript levels of the identified proteins in both organs. In doing so, they identified 13 SFPs that were present in both seminal vesicles and spermathecae. These transferred SFPs included heat shock proteins, protease inhibitors, an angiotensin-converting enzyme (TcACE), and three further proteins that had not previously been identified in the seminal fluids of other insects. Next, they individually knocked down their expression by injecting gene-specific dsRNA into male pupae. Injected males were then mated with virgin females and the number of eggs laid was determined and compared with that of matings with males who had normal levels of SFPs.
The gene that showed the most significant effect on female reproduction was TcACE. Its knock-down in males caused a significant reduction in egg production by females. To exclude the possibility that the observed effect on egg production is caused by a reduced number of sperm, the team injected dsRNA into pupae and determined the sperm number in testes prepared later from 5 day old adult males. The knock-down of TcACE expression did not affect sperm numbers, although TcACE transcript levels were significantly decreased in the testes and total SFP amounts were diminished. However, sperm quality was affected, because there was a higher portion of abnormal sperm in the testes of dsRNA-injected males, suggesting that TcACE protects sperm in the seminal fluid. Finally, the scientists also examined TcACE gene expression in females. They demonstrated that TcACE is expressed not only in the male reproductive tract but also in spermatheca, suggesting that females sustain sperm protection during storage by continuing TcACE production. Speculating about TcACEs' molecular role in T. castaneum, the authors point out that angiotensin-converting enzymes have been detected in seminal fluids from insects to humans. As these highly conserved enzymes are known to convert hormone precursors into their active forms, TcACE may be involved in processing peptide hormones that protect sperm.
By using the powerful RNAi technique in the model beetle T. castaneum, Palli and his colleagues have provided convincing evidence that TcACE plays an important role in protecting the sperm in the seminal fluid during transfer and in the spermatheca during storage.